The effect of dentifrice ingredients on enamel erosion prevention and repair

Fluoride is a key dentifrice ingredient for mitigating dental erosion and promoting remineralisation in tooth enamel. A dentifrice formulation (NaF/CL ‐ where C = Copolymer, L = Lactate), optimised for fluoride delivery, containing sodium fluoride, polyvinylmethylether–maleic anhydride (PVM/MA) copolymer and lactate ion at controlled pH of 6.2 is compared with six commercial dentifrices from European and US regulatory regions. The in vitro study utilised white light interferometry (WLI) and dynamic secondary ion mass spectrometry (DSIMS) to assess dental erosion resistance and remineralisation potential of the dentifrices. For WLI, polished enamel samples were immersed in dentifrice slurry (1:3 wt./wt. in artificial saliva, 2 min), brushed, washed in deionised water, acid challenged (1% citric acid, pH 3.8, 5 min), washed and air dried. Surface roughness and bulk tissue loss were measured. DSIMS samples for fluoride uptake were acid challenged (1% citric acid, pH 3.8, 5 min), rinsed, immersed in dentifrice slurry (1:3 wt./wt. in artificial saliva, 2 min), washed and air dried. DSIMS samples for 44Ca uptake were prepared similarly but with 44Ca‐doped artificial saliva. The NaF/CL dentifrice provided highest protection against dental erosion, with highest fluoride and 44Ca uptake in all treatment groups (n = 5 per group). Dentifrices containing other fluoride salts and/or ingredients known to inhibit fluoride uptake (e.g., polyphosphates, sodium lauryl sulfate), performed significantly worse.


| INTRODUCTION
The loss of human tooth enamel through wear can occur by three defined processes, that is, dental erosion, dental attrition and dental abrasion. 1 Dental erosion involves the chemical loss of mineralised tooth substance caused by exposure to acids not derived from oral bacteria, that is, from stomach acids or food and drink. This results in softening and removal of enamel across the whole tooth surface. 2 The process typically extends from 0.1 μm depth in the early stages of erosion to beyond 100 μm after prolonged exposure. [3][4][5][6] In dental attrition the physical loss of mineralised tooth material is caused by tooth-to-tooth contact, for example, tooth grinding (known as bruxism), whereas dental abrasion is caused by contact with objects other than teeth. Typical examples of dental abrasion are the effects of chewing certain foods or tooth brushing.
Concern over the increasing prevalence of dental erosion has been growing for more than 20 years, 7 and it has recently been described as 'an increasingly relevant problem'. 8 Dental erosion is distinct from dental caries, which has a tendency to form in localised and relatively inaccessible areas of the teeth when food containing sugars or starch is converted to acids by dental plaque bacteria present on the tooth surface. Fluoride has been shown to be effective at inhibiting dietary acid-mediated erosion of dental hard tissues as well as promoting repair of demineralised incipient erosive lesions, that is, 'remineralisation'. [9][10][11] In vitro studies, primarily aimed at investigating caries prevention, have also shown that adequate availability of calcium, phosphate and fluoride ions can produce significant remineralisation of lesions in enamel. 12,13 The challenge, therefore, is to design delivery systems which provide sufficient quantities of these active ions to optimise remineralisation.
Fluoride is clearly a key ingredient in dentifrices for mitigation of dental erosion and promotion of remineralisation. However, dentifrices are complex vehicles for therapeutic agents, and several ingredients commonly found in formulations can interfere with fluoride action. This may occur by direct precipitation of fluoride ion via certain polyvalent metal ions; reduction of fluoride binding to enamel surfaces by ionic surfactants, for example, sodium lauryl sulfate; 14 or interference with fluoride-promoted remineralisation processes by polyvalent metal ions and polyphosphates. 15,16 It is not possible to simply add more fluoride to formulations to offset these losses because national and international regulatory bodies set limits to dentifrice fluoride content in order to avoid harmful over-exposure from ingested product. Hence, the avoidance of fluoride bioactivity loss is critical.
One of the dentifrice formulations used in this study, Pronamel, was developed over a decade ago to avoid the use of the interfering ingredients described above. 16 In this study the established formulation technology has been further developed to include agents which have been found to be able to promote fluoride's action on enamel. Specifically, the combination of polyvinylmethylether-maleic anhydride (PVM/MA) copolymer and lactate ion (as sodium lactate), with the formulation pH controlled to 6.2 (designated as NaF/CL in this study), has been found to increase fluoride uptake and enhance fluoride-mediated acid resistance. 17 This modified formulation has the commercial name Pronamel Intensive Enamel Repair. In this study the two formulations were initially compared using standard US Federal Drug Administration (FDA) assays, that is, enamel fluoride uptake (EFU) 18 and enamel solubility reduction (ESR), 19 in order to assess the potential benefits.
With this background of varying dentifrice formulations and constant iterative improvements there is an associated analytical challenge to apply methods which adequately monitor the effects of erosion, the ingress of any beneficial agents into enamel surfaces and the progress of remineralisation on the tooth surface. The initial prerequisite for such experiments is to obtain a set of human tooth enamel samples which are effectively identical in terms of physical uniformity and composition. Hence, all enamel samples are subjected to an identical cleaning and polishing procedure designed to remove surface debris and any fluoride in the enamel from water fluoridation and/or toothpaste use.
Over the past few decades a number of analytical techniques have been developed and utilised for the assessment of the chemical composition and physical structure of tooth enamel lesions. These techniques have been reviewed extensively [20][21][22] and include standard radiography, 23 transverse micro-radiography (TMR), 24 secondary ion mass spectrometry (SIMS), 25 electron probe micro-analysis (EPMA), 26 surface microhardness (SMH) and cross-sectional microhardness (CSMH). For erosion studies, SMH has been consistently used to characterise mineralisation status and surface strength, with profilometrybased techniques also providing further physical information on topography and erosion wear depth. [27][28][29] In a series of studies the present authors have used a combination of white light interferometry (WLI) to measure bulk tissue loss through erosive challenges and dynamic secondary ion mass spectrometry (DSIMS) to compare uptake of fluoride, from various dentifrice treatments, into lesioned enamel. 30,31 WLI provides measurement of surface roughness and tissue loss in the z plane (depth) on the nanometer to tens of microns scale, whereas DSIMS allows acquisition of chemical images from cross sections of treated enamel with sub-micron spatial resolution and detection sensitivities on the ppm-ppb scale for, for example, fluoride. Retrospective line-scan analysis from DSIMS allows reconstruction of in-depth relative concentration profiles, for example, for assessment of relative fluoride uptake.
A significant challenge arises when the purpose of the investigation is to monitor chemical compositional changes of remineralised lesions, because the remineralising agents (i.e., calcium and phosphate) are chemically indistinguishable from the enamel substrate. In order to overcome this challenge, 44 Ca-labelling of calcium phosphate in a dentifrice has been used to successfully demonstrate uptake of 'new' calcium into demineralised enamel. 32 In more recent remineralisation studies, the present authors have used isotopically enhanced calcium chloride containing 97% 44 Ca in the remineralising artificial saliva medium. 33 Measurement of the cross-sectional distribution of calcium isotopes (e.g., 42 Ca, 44 Ca) by DSIMS imaging, following treatment of enamel with 44 Ca-labelled artificial saliva, provides clear distinction between 'original' calcium and 'new' calcium. It is clear, therefore, that WLI and DSIMS enable the quantification and visualisation of the effects of different toothpaste formulations on fluoride uptake, remineralisation and protection against demineralisation.
The main aim of this study was to assess the effects of different dentifrice formulations, marketed in European and US regulatory regions, by measuring relative fluoride uptake, surface roughening due to demineralisation, bulk tissue loss after brushing and uptake of new calcium-that is, remineralisation-using artificial erosive lesions in human enamel in vitro. A range of commercial dentifrices were placed into three groups, that is, two groups from the European regulatory region (EU1 and EU2, where the upper limit on fluoride content is 1500 ppm F) and one group from the US regulatory region (US, where the standard fluoride level is 1100-1150 ppm F). Each group included a fluoride-free control. The ability of the dentifrices to suppress enamel surface roughening due to demineralisation and bulk tissue loss (after brushing) was assessed using WLI. DSIMS was used to measure the relative uptake of fluoride and calcium, in separate experiments.

| Pronamel formulations
A summary of the dentifrices used in the initial assessment of the two Pronamel formulations is shown in Table 1. is quantified as the amount of phosphate dissolved from the enamel surface, measured using photoelectric colorimetry. 19 The reduction in mineral loss due to treatment is calculated as a percentage (i.e., [mineral loss post-treatment/mineral loss pre-treatment] × 100).

| EFU and ESR assays
Hence, the EFU is a measure of the ability of the product to deliver the active ingredient (fluoride) to enamel surfaces, and the ESR is a measure of the ability of the delivered active ingredient to protect those surfaces from acid exposure.

| Commercial dentifrices
The commercial dentifrices used in the WLI and DSIMS studies are shown in Table 2 below.

| Enamel sample preparation
Human tooth enamel samples were randomly selected and embedded in 5 mm thick acrylic resin blocks. All enamel samples were polished with progressively finer silicon carbide paper before a final polishing stage with P1200 and P2400 grit silicon carbide paper. This procedure is designed to provide a smooth, flat, uniform surface on which to perform studies and to remove surface debris and any fluoride already present in the enamel from water fluoridation and/or prior toothpaste use. Ca isotopically enriched calcium chloride was also obtained from Sigma-Aldrich UK.

| WLI
Prior to any treatment regime, an erosion window was created on each resin-mounted human enamel specimen by placing acid-resistant adhesive tape across the enamel surface, leaving approximately 50% of the enamel surface area protected. For each of the three studies, specimens were divided into four treatment groups (n = 5) and immersed into one of the dentifrice slurries (1:3 wt/wt in artificial saliva, 2 min) followed by controlled manual brushing for 2 min before washing for 1 min with deionised water. All specimens were then subjected to an acid challenge by suspension in 1% citric acid, pH 3.8 for 5 min, without agitation. Finally, specimens were washed with deionised water and air dried.
Prior to WLI analysis, the position of the protective tape was marked on each sample and then the tape carefully removed to expose a non-eroded reference region, immediately adjacent to the A MicroXAM (ADE phase shift) white light interferometer was used to acquire topographic data. Each enamel sample was initially scanned over three areas of 687 μm × 511 μm containing exposed/treated and tape-protected regions, to determine the step heights. This was followed by scanning three areas of 687 μm × 511 μm within the exposed/treated region for surface roughness determination. A Z (height) range of up to 300 μm was used for the measurements. Proprietary image analysis software (SPIP™-Image Metrology A/S) was then used to process the raw data to produce images and surface roughness parameters. In each area, void pixels were removed and outlier spikes filtered.
For the roughness analysis areas, a 2 polynomial plane correction was applied to remove gross form (curvature) and allow accurate roughness measurements.
For the step height analysis areas, the manual tilt function was used to maximise the flatness of the reference area. An averaged line scan was used across the interface between the treated and untreated regions to determine the step height.

| 44 Ca uptake studies
For each of the three 44 Ca uptake studies, 20 polished enamel samples were initially subjected to an acid challenge by suspending in 1% citric acid, pH 3.8 for 5 min, without agitation. After washing with deionised water, specimens were then divided into four treatment 3 | RESULTS

| EFU and ESR
The results of the comparison of EFU and ESR values for the NaF and NaF/CL formulations, compared to a fluoride-free placebo, are presented in Table 3.
It is evident that both fluoride uptake and enamel protection are enhanced in NaF/CL. This was confirmed by statistical analysis to be significant at greater than 95% confidence in both cases.
On the basis of the positive benefits offered by NaF/CL in the EFU and ESR data, this modified formulation was included in the three study groups for comparison with other commercial dentifrices using the WLI and DSIMS techniques.

| Surface roughness and bulk tissue loss by WLI
Mean surface roughness measurements of brushed/acid-challenged regions of each of the four treatment groups (n = 5) are presented in Figure 1 for the EU1, EU2 and US studies.  Figure 3 where representative 3D images from each of the four treatment groups are shown.  In the EU2 study, NaF/CL provides a fluoride uptake 2 times higher than NaF/Pyr/SLS (EU) and 60 times greater than ZnHA For the US study, NaF/CL has the highest fluoride uptake within the four treatment groups, that is, 5 times higher uptake compared to NaF/Pyr/SLS (US) and 38 times higher F uptake c.f. SnF 2 /SLS/Zn.
The inter-group differences in F uptake are all statistically significant at 95% confidence level with p values all less than 0.05.
A series of representative DSIMS image overlays are presented in Figure 5 for the EU1 study group.
In each image, fluoride uptake is clearly shown by the designation of red colouration to F with the tooth enamel substrate represented by O signal and shown in cyan.  44 Ca uptake integrals for each treatment group within the three studies are plotted in Figure 6.

| 44 Calcium uptake by DSIMS
In the EU1 and EU2 studies, NaF/CL consistently exhibited the highest level of 44 Figure 7 for the EU2 study.
In each of the images 44

| DISCUSSION
It has already been established that fluoride is a key ingredient in dentifrices for mitigation of dental erosion by inhibition of demineralisation and promotion of remineralisation. The delivery of fluoride to the tooth surface from a dentifrice is optimal when ingredients which interfere with fluoride delivery are removed from formulation 16 and agents which can increase uptake of fluoride are present. 15,17 In this study two key aspects of dentifrice development have been addressed, that is, iterative formulation improvement and a comparison of a range of competitive formulations which have been designed to provide improved resistance to dental erosion through enhanced fluoride uptake and repair demineralised enamel through remineralisation.
A recent clinical study, in which 'in situ' enamel specimens were mounted in an intra-oral appliance, 15  In order to assess comparative resistance to dental erosion provided by the dentifrices, enamel specimens were subjected to a dentifrice treatment/brushing/acid challenge cycle, followed by mea-  From the results within the two parts of this study, it is clear that formulation ingredients can have a significant impact on dentifrice performance in terms of protection against dental erosion by fluoride uptake, promotion of remineralisation and protection against demineralisation. In the first part of the study, one aspect of the potential improvement of the NaF/CL formulation, c.f. NaF, was the control of pH. It has been known for many years that a limited lowering of the pH of fluoride treatments 34 increases fluoride uptake to enamel without leading to demineralisation. 35 It has also been suggested to have the potential to enhance caries protection from toothpastes. 36 38,39 In the NaF/CL formulation it is apparent that the protective effect of the polymer system has been achieved without compromising fluoride uptake or remineralisation.
All of the dentifrices tested within each of the WLI and DSIMS study groups, that is, EU1, EU2 and US groups, were formulated with the same or very similar fluoride concentrations. It is therefore reasonable to assume that the measured differences in fluoride uptake and any ensuing benefits must be related to the presence of ingredients which either restrict or promote F uptake or to mechanistic differences in the delivery of free fluoride ion from solution to the enamel surface. With the exception of NaF/CL, all other commercial dentifrice formulations contained ingredients which have been found to either limit fluoride uptake, remineralisation or both, that is, sodium lauryl sulfate -SLS 14 , polyphosphates [40][41][42] and polyvalent metal ions. 17,43,44 The inhibitory effects displayed by these agents are believed to be related to their affinity for enamel surfaces. Sodium lauryl sulfate has been shown to inhibit uptake of fluoride into enamel from alkali-soluble sources such as sodium fluoride. 14 29,47,48 The balance of these positive and negative effects will determine the overall protective effect of the individual formulation.
Finally, the form of fluoride used is also important to its protective effect against dental erosion. It would be reasonable to assume that formulations containing sodium fluoride and stannous fluoride will have essentially all fluoride available as free fluoride ion in solution under the experimental conditions used in this study. However, it is notable that the stannous fluoride-containing formulations, SnF 2 / HMP in the EU1 study and SnF 2 /SLS/Zn in the US study, were ranked below NaF/CL in the mean surface roughness/bulk tissue loss measurements by WLI but performed relatively poorly in terms of fluoride uptake (and consequently 44 Ca uptake) measured by DSIMS. It is known that stannous fluoride treatment results in formation of a protective barrier layer on the enamel surface but the exact nature of that layer is not yet clear. 49,50 It has also been suggested that The dentifrice containing SMFP (SMFP/CS in the EU1 study) showed significantly lower performance in surface roughness, bulk tissue loss, fluoride uptake and 44 Ca uptake compared to NaF/CL. The release of free fluoride ion from SMFP will depend on cleavage of the covalent bond between phosphate and fluoride by enzymatic hydrolysis during treatment. In the mouth, this hydrolysis occurs mainly by the action of plaque bacteria. 52 Although this mechanism is relevant to demineralisation due to caries which occurs under plaque-coated surfaces, it is of little relevance to dental erosion which occurs principally on exposed surfaces with little or no plaque. 53 It is also known that the presence of sodium lauryl sulfate may inhibit this hydrolysis process when present in SMFP formulations. 54 Hence, it is not surprising that release of fluoride ion-mediated effects were significantly lower in SMFP/CS compared to the NaF/CL formulation.

| CONCLUSIONS
This study has shown the significant improvement in EFU and reduction of enamel solubility achieved by the addition of PVM/MA copolymer and sodium lactate at pH 6.2 (NaF/CL), to an established sodium fluoride-based dentifrice formulation (NaF). Comparisons of the NaF/CL formulation were undertaken with a range of commercial dentifrices from the European and US regulatory regions, which contained different fluoride sources and formulation ingredients, measuring (i) surface roughness and bulk tissue loss by WLI after a treatment/brushing/acid challenge cycle and (ii) fluoride uptake and 44 Ca uptake after acid challenge/dentifrice treatment cycles by DSIMS.
These in vitro studies showed that the NaF/CL formulation provided superior performance in promoting remineralisation and protecting against dental erosion.